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Nitric Oxide (NO) has been shown to be a key regulator of myocyte contraction. NO is produced endogenously via two NO synthase (NOS) isoforms: NOS1 and NOS3. In disease, NO signaling becomes altered leading to contractile dysfunction. In order to understand NO signaling in disease, it is imperative to determine how different NOS isoforms regulate contraction. At present, NOS3’s role in contraction is known. However, it remains unclear how NOS1 regulates contraction. Thus, the purpose of this thesis was to determine NOS1’s role in myocyte contraction.We studied the role of NOS1 signaling on myocyte contraction in NOS1 Knockout (KO) trabeculae and myocytes. In both models, we observed decreased contraction and prolonged relaxation vs. WT. Due to the fact relaxation is prolonged in NOS1KO myocytes, we determined if NOS1 regulated SR Ca2+ handling. Specifically, NOS1KO myocytes exhibited decreased serine 16 PLB phosphorylation (PLB-P) and SR Ca2+ load compared to WT. We next determined if the effects of NOS1KO could be reversed by via β-AR receptor stimulation. NOS1KO myocytes exhibited a decreased functional response to β-AR receptor stimulation compared to WT myocytes. However, β-AR receptor stimulation abolished the prolonged relaxation observed in NOS1KO myocytes. These data suggest NOS1 regulates PLB and β-AR receptor stimulation can abolish the prolonged relaxation observed in NOS1KO myocytes.NO can signal either via cyclic-GMP or independently of cyclic-GMP. Therefore, we investigated the signaling pathway thru which NOS1 signals. ODQ (guanylate cyclase inhibitor) had no effect in WT myocytes. Conversely, FeTPPS, a peroxynitrite decomposition catalyst, produced contractile dysfunction and decreased serine 16 PLB-P in WT myocytes similar to that observed in NOS1KO myocytes. These data suggest NOS1 signals in a cyclic-GMP independent manner via formation of peroxynitrite.Nitroso-redox imbalance has been observed in NOS1KO myocytes, and has been shown to play a role in the contractile dysfunction present in NOS1KO myocytes. Therefore, we next determined if a novel superoxide scavenger, EMEPO, was able to correct this nitroso-redox imbalance, thus improving contraction in NOS1KO myocytes. EMEPO normalized superoxide and NO levels in NOS1KO myocytes to the level of WT. Furthermore, we investigated the effect of EMEPO on myocyte contractile function. EMEPO increased myocyte contraction and accelerated the rate of relaxation via regulation of SR Ca2+ handling. Specifically, EMEPO increased RyR activity and serine 16 PLB-P in NOS1KO myocytes. Interestingly, EMEPO’s effects on contraction in NOS1KO myocytes were significantly greater than those of a superoxide scavenger or a NO donor. In addition, EMEPO potentiated the functional response of NOS1KO myocytes to β-AR receptor stimuli. These data suggest EMEPO is a novel superoxide scavenger that improves contractile function via regulation of SR Ca2+ handling.The results presented herein provide evidence that NOS1 signaling leads to positive inotropy and lusitropy in the heart. In addition, it appears EMEPO, a novel superoxide scavenger, can restore nitroso-redox balance under conditions of oxidative stress. Furthermore, the present work may have therapeutic implications for the treatment of cardiovascular disease, a pathological state in which NO signaling is altered. |
